Molecular Biology 0202301 PDF

Molecular Biology 0202301 Medical Laboratory Science Department Week 09: Pulsed-Field Gel Electrophoresis (PFGE) Textbook: - Robert F. Weaver, Molecular Biology, 5th edit...

Molecular Biology 0202301 Medical Laboratory Science Department Week 09: Pulsed-Field Gel Electrophoresis (PFGE) Textbook: - Robert F. Weaver, Molecular Biology, 5th edition - Watson J, et al. (2014). Molecular Biology of the Gene, 7th edition  Pulsed-Field Gel Electrophoresis (PFGE) 2 Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024  Electrophoresis Ladder 100 500 1000 3000 5000 10000 15000 20000 30000 10000 5000 3000 1000 PFGE 500 100 4 Objectives To explain how the Pulsed-Field Gel Electrophoresis (PFGE) can be constructed Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 5 Pulsed-Field Gel Electrophoresis (PFGE) - Standard gel electrophoresis is unable to separate very large (higher than 20-30 kb) molecules of DNA effectively. - DNA molecules larger than 15-20kb migrating will move together in a size-independent manner. - Alternating voltage gradient to improve the resolution of large molecules - This technique is known a Pulsed-Field Gel Electrophoresis (PFGE) Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 6 Pulsed-Field Gel Electrophoresis (PFGE) More than one alternating electric field to a gel matrix - PFGE resolves DNA by alternating the electrical field between spatially distinct pairs of electrodes. - This technique results in the separation of DNA fragments of up to ~10 Mb by their reorientation and movement at different speeds through the pores of an agarose gel. Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 7 PFGE: Principle Smaller Changes Faster - When the electrical field is applied to the gel, DNA segments elongate; DNA relaxation depends on fragment's size. - The first electrical field is then switched to the second field according to the run specifications. Then, the DNA must change conformation again and reorient before it can migrate in the direction of this field. Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 8 PFGE Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 9 Pulsed-Field Gel Electrophoresis (PFGE) Used in conjugation with Rare-Cutter Restriction Endonucleases: - Recognize CG islands in genomic DNA of vertebrates (e.g: NotI recognizes GCGGCCGC) - CG islands occur at low frequency in vertebrates - Few recognition sites, the result is small number of large fragments - Treated and separated to generate a DNA fingerprint Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 10 PFGE – Restriction Enzyme The choice of the restiction enzyme is based on the GC% – The goal is to have a limited number of bands (10 – 20 bands). Restriction Enzyme Organism Enteroccocus Staphylococcus Listeria Streptococcus Clostridium Campylobacter Sma I Not I Escherichia coli Helicobacter pylori Neisseria meningitidis Bacteroides Vibrio cholerae Pseudomonas Neisseria gonorrheae Burkholderia cepacia Serratia marcescens Enterobacter Spe I Sfi I Legionella pneumophila Proteus mirabilis Corynebacterium diphtheriae Xba I E. coli O157: H7 Salmonella Klebsiella Shigella Yersinia pestis Mycobacterium Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 11 PFGE – Program examples Switch time (sec) (kb) Angle (°) Gradient Duration Strain (V/cm) (h) Initial Final P. aeruginosa 5,3 34,9 50-500 120 6,0 20,0 E. coli 5,3 49,9 50-700 120 6,0 20,0 Enterobacter 5,3 34,9 25-300 120 6,0 20,0 Candida 90 325 1000-3500 120 6,0 48,0 E. coli O157:H7 2,2 54,2 50-500 120 6,0 22,0 C. diphtheriae 1,0 17,0 25-300 120 6,0 18,5 Mycobacterium 1,0 23,0 25-400 120 6,0 18,5 Salmonella KS 5,3 49,9 50-700 120 6,0 19,7 Helicobacter pylori 5,3 60,0 50-900 120 6,0 19,5 Salmonella & H. pylori 5,3 66,0 50-1000 120 6,0 19,5 Candida glabrata 5,3 120,0 50-2000 120 6,0 19,5 Candida glabrata 120,0 600,0 1000-3000 106 3,5 48,0 Candida krusei 600,0 1800,0 1000-4500 106 2,3 72,0 E. coli O157:H7 CDC 2,2 54,2 / 120 6,0 22,0 Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 12 Restriction Mapping - Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. - Restriction mapping requires the use of restriction enzymes. Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 13 PFGE: Protocol - Collect the sample - Load to agarose suspension (Similar to Gelatin) - Release the DNA (Cell lysing; protein digestion and RE) - Load the agarose (Including DNA) to the gel - Electrophoresis - Generate a DNA fingerprint Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 14 PFGE steps Agarose into the plugs Cell Suspension/ Agarose / Plugs Cell Wall destruction Lysozyme * (or other ) / 1Hr / 37°C Protein digestion Proteinase K /Over Night / 50°C / Washes before restriction DNA Resriction Restriction enzyme / Over Night / 25 or 37°C / 20 - 50 U/insert Migration TBE 0.5X or TAE 1X / 18 – 24 Hrs / 14°C Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024  Agarose Cell suspension Lysing RE Load to Gel 15 PFGE Protocol Restriction Mapping DNA fingerprint Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 16 Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 17 PFGE Result Analysis: Strain clonality detrmination Statistical form: Clonality higher or equal to 70-80% between the strains Graphical form: Dendograms creation (Phylogenetic trees) No. of genetic differences Category compared with outbreak strain Interpretation Genetic event Fragments Indistinguishable 0 0 Isolate is part of the outbreak Closely related 1 2-3 Isolate is probably part of the outbreak Possibly related 2 4-6 Isolate is possibly part of the outbreak Isolate is not part of the outbreak Different >3 >7 Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 18 Phylogenetic trees Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 19 PFGE: Applications - Genotyping or genetic fingerprinting - Epidemiological studies of pathogenic organisms - Identification of organism/pathogens isolated from environmental or clinical samples - Identification of antibiotics resistant strains - Subtyping among strains; Phylogenetic analysis and Taxonomy Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 20 PFGE: Affected by…… - Voltage (V/cm): ~ 5-7 V/cm ( 200V/ ~33cm) - Angel - Switching Time: Longer  Further Migration of large segments - Temperature: Chiller!!!!!! Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 21 Effects of Buffer Temperature Electrophoresis Buffer can be chilled Higher the temperature: Faster is the migration - The resolution decreases with the temperarture increase - Buffer chilled at +4°C gives the best results - The most common is to chilled the buffer between 12 to 15°C Best compromise between resolution and migration time Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 22 Advises & Troubleshooting Optimization at several steps - Plug preparation - Lysis step Important to « Standardize » - Restriction step - Running conditions 3 Possibles scenarios: I. NO band at all on all the tracks of the gel (Samples /Control Strain/MW) II. NO band for all the samples and the control strain but good result for the MW III. NO band for all the samples but good result for the control strain and the MW Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 23 Advises & Troubleshooting NO bands for All the tracks NO band for all the Samples NO band for all the Samples (Samples/Control/MW) and the Control Results but good for the MW but good result for Control and MW - Sample preparation - Sample preparation - Sample preparation - Lysis - Lysis - Lysis Conclusion - Restriction - Restriction - Running conditions Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 24 Conclusion PFGE is a reliable technique that needs attention on the following steps: - Type of the organism: Reagent change ( e.g. Restriction Enzyme) - Adding a Control Strain - Work with sterile material (For all the steps) - Avoid powdered gloves - The quailty of water for the buffer preparation Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 25 See YOU Next Lecture  Molecular Biology_Week09- Pulsed-Field Gel Electrophoresis (PFGE) Saturday, November 16, 2024 Molecular Biology 0202301 Medical Laboratory Science Department Week 10_Part I: Probes & Hybridization Textbook: - Robert F. Weaver, Molecular Biology, 5th edition - Watson J, et al. (2014). Molecular Biology of the Gene, 7th edition 2 Objectives I. To know the definition of hybridization and probes II. To know the type and the method of probes labeling III. To know the applications of probes and hybridization Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 3 Nucleic acid Probes - A probe is a Synthetic single-stranded Oligonucleotides sequence of DNA or RNA used to search for its complementary sequence in a sample genome. - This sequence is complement to Part or All of target sequence - Labeled with a marker; useful for Identification & Quantitation Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 4 Sunday, November 24, 2024 Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 5 Probes Types of Labels: I. Radioactive: (e.g. 32P, 35S, 14C, 3H) II. Fluorescent: (e.g. DiG, Biotin, FAM) III. Biotinylated: (e.g. avidin-streptavidin) Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 6 Labeling Methods I. Strand Synthesis: PCR to Insert Labeled Nucleotides into Probe - Template (DNA or RNA): Generate Labeled Fragments - One of Labeled dNTPs (e.g. 32P- dCTP) A. Nick-Translation B. PCR-Mediated Labeling II. End-Labeling: γ-Phosphate from 32P-ATP to the 5’-OH - T4 Polynucleotide Kinase Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 7 Strand Synthesis: Nick-Translation I. DNase I randomly nicks double-stranded DNA breaking a phosphodiester bond to leave 5′ phosphate termini. II. The Escherichia coli DNA pol-I I activities: - 5′ to 3′ polymerase activity that requires a single-stranded template and a primer with a 3′ hydroxyl group to synthesize a new nucleotide chain complementary to the template (Labeled Nucleotide) - 5′ to 3′ exonuclease activity that degrades double-stranded DNA from a free 5′ end Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 8 Strand Synthesis: PCR-Mediated Labeling - Simultaneous amplification and labeling of DNA probes - In the presence of appropriately labeled nucleotides, PCR products are labeled as they are being synthesized. Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 9 End-Labeling: T4 Polynucleotide Kinase - The DNA 5′ End-Labeling System is a complete system for phosphorylating both oligonucleotides and DNA fragments - Using T4 polynucleotide kinase (PNK), which catalyzes the transfer of the terminal [γ-32P]phosphate of ATP to the 5′-hydroxyl terminus of a DNA molecule. Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 10 Nucleic Acid Hybridization (NAH) Formation a duplex between TWO complementary sequences by means of hydrogen bonding between base pairs. - DNA-DNA - DNA-RNA - RNA-RNA Identify how much the DNA molecules are close Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 11 Factors Affecting Hybridization - Strand Length - Base Composition: % GC - Probe Mismatch - Temperature & Melting Temperature - Chemical Environment: - Monovalent cations: Stabilize the Duplex - Polar Molecules: Denaturat - Hybridization Stringency: - High NaCl/Low Temp: Low Stringency - Low NaCl/High Temp: High Stringency Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 12 Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 13 Application of Hybridization Filter Hybridization: Most Common Applications - Southern Blots - Northern Blots - Dot/Slot Blots - Colony Hybridization In-Silica Hybridization - In Situ Hybridization (Tissue) - Chromosomal (FISH) - Microarrays Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 14 See YOU Next Lecture  Molecular Biology_Week10-Part-I_Probes & Hybridization Sunday, November 24, 2024 Molecular Biology 0202301 Medical Laboratory Science Department Week 10_Part II: Hybridization_Filter Hybridization Textbook: - Robert F. Weaver, Molecular Biology, 5th edition - Watson J, et al. (2014). Molecular Biology of the Gene, 7th edition 2 Objectives To know the definitions and the protocols of the different Filter Hybridization techniques Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024 3 Application of Hybridization Filter Hybridization: Most Common Applications - Southern Blots - Northern Blots - Dot/Slot Blots - Colony Hybridization In-Silica Hybridization - In Situ Hybridization (Tissue) - Chromosomal (FISH) - Microarrays Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024 4 Hybridization: Southern & Northern Blot Labeled Probe Hybridizing to Fractionated and Immobilized: - DNA (Southern) - RNA (Northern) DNA/RNA Separation by Size  Identifying the Fragment Denaturation DNA & Transferring from the gel to a solid support filter Hybridizing with a complementary Nucleic Acid Probe Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024 5 Southern/Northern Blot: Protocol I. Restriction digestion and agarose gel electrophoresis II. DNA denaturation (NaOH: Permit binding to the filter) and transfer to filter paper (Capillary flow) III. Hybridization to probe and visualization Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024  Restriction digestion and electrophoresis 6 Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024  Denaturation and transfer to filter paper 7 Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024  Hybridization and visualization 8 Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024  Southern Blot Result 9 Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024 Southern Blot 11 Hybridization: Dot-Blot - Allele-Specific Oligonucleotides (ASO) Probes - Labeled probes are hybridized to immobilized target genomic DNA - DNA/RNA are bound directly to a solid support filter - No size separation - Ideal for quantitative measurements Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024 12 Hybridization: Dot-Blot - DNA samples are spotted on a filter in dots of uniform diameter. - The filter is subject to denaturing conditions and then hybridized with a probe. - The advantage: Can be applied to a large number of DNA samples being fast and cheap - no electrophoresis and DNA transfer are required Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024  Hybridization: Colony Hybridization 13 Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024 14 See YOU Next Lecture  Molecular Biology_Week10-Part-II_Hybridization_Filter Hybridization Sunday, November 24, 2024